Divergent Physiological Functions of Four Atg22-like Proteins in Conidial Germination, Development, and Virulence of the Entomopathogenic Fungus Beauveria bassiana

In yeast, Atg22 functions as a vacuolar efflux transporter to release the nutrients from the vacuole to the cytosol after the degradation of autophagic bodies. There are more than one Atg22 domain-containing proteins in filamentous fungi, but their physiological roles are largely unknown. In this study, four Atg22-like proteins (BbAtg22A through D) were functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. These Atg22-like proteins exhibit different sub-cellular distributions. BbAtg22A localizes in lipid droplets. BbAtg22B and BbAtg22C are completely distributed in the vacuole, and BbAtg22D has an additional association with the cytomembrane. The ablation of Atg22-like proteins did not block autophagy. Four Atg22-like proteins systematically contribute to the fungal response to starvation and virulence in B. bassiana. With the exception of ∆Bbatg22C, the other three proteins contribute to dimorphic transmission. Additionally, BbAtg22A and BbAtg22D are required for cytomembrane integrity. Meanwhile, four Atg22-like proteins contribute to conidiation. Therefore, Atg22-like proteins link distinct sub-cellular structures for the development and virulence in B. bassiana. Our findings provide a novel insight into the non-autophagic roles of autophagy-related genes in filamentous fungi.


Introduction
Beauveria bassiana acts as an important representative entomopathogenic fungus and has been widely considered in the biocontrol of insect pests [1,2]. In eukaryotes, autophagy is a conserved process for recycling macromolecules and organelles, which are degraded in vacuole/lysosome [3]. In B. bassiana, this process is associated with the entire infection cycle and plays essential roles in the fungal stress response, development and virulence [4,5]. However, the roles of the autophagic process remain largely unknown in the insect pathogenic fungi.
At present, 43 autophagy-related genes (ATGs) have been identified and functionally characterized in yeast [6,7], in which 18 ATGs (e.g., ATG1 and ATG8) are considered as 'core' machinery genes indispensable for the autophagic process [8]. In B. bassiana, Atg1 (a serine/threonine protein kinase) are indispensable for autophagosome induction and expansion [4,9]. The components in the ubiquitin-like conjugation system (e.g., Atg3, Atg5, Atg7, and Atg8) are absolutely necessary for autophagy [4,[10][11][12]. In addition, Atg1 directly phosphorylates the E2-like enzyme Atg3 of the ubiquitin-like conjugation system, which is indispensable for the functionality of this conjugation system [9]. Atg11 acts as an essential scaffold protein and mediates the selective degradation of mitochondria and peroxisomes [13]. In B. bassiana pexophagy, peroxisomes are labeled by pexoxin 14, which is recognized by Nbr1 (neighbor of BRCA1 gene 1). Then, Nbr1 recruits the targeted peroxisomes into the autophagosomes via the direct interaction with Atg8 [5]. Among the 'core' ATGs, the genes involved in the degradation and transportation system (DTS) differ in some degrees among fungal species. For example, the number of Atg22 significantly varies among different fungi, but at least one Atg22 is present in a fungus [6]. In yeast cells, Atg22 function as a permease responsible for the transportation of degradation products from the vacuole to the cytosol. In detail, Atg22 has functional overlaps with two other vacuolar amino acid effluxers, Avt3 and Avt4. After autophagic degradation, Atg22 mediates the efflux of leucine and other amino acids resulting from the vacuoles. The recycled amino acids maintain protein synthesis and cell viability under nitrogen starvation [14]. The homolog survey indicates that there are four Atg22-like proteins in B. bassiana, which is not prevalent among fungal species [6]. This implicates that B. bassiana might use a set of Atg22-like permeases at different stages of its lifecycle, and the action mode of Atg22-like proteins needs to be revealed at the molecular levels.
In this study, four Atg22-like proteins were functionally characterized in B. bassiana via the construction gene disruption and complementation mutant strain. Four proteins displayed divergent sub-cellular localizations and biological functions, including fungal differentiation, stress resistance and virulence.

Bioinformatic Analyses and Sub-Cellular Localizations of Atg22-like Proteins in B. bassiana
Four Atg22-like proteins have been recognized in the B. bassiana genome [6]. Their domain architectures were analyzed through the online portal, SMART (http://smart. embl-heidelberg.de, accessed on 15 December 2022) [15]. Phylogenetic and molecular evolutionary analyses for the Atg22-like proteins were conducted using MEGA version 5. Relationships among the Atg22-like proteins were constructed using the Neighbour joining method and the bootstrap values generated from 1000 replicates [16].
Cellular localizations of the Atg22-like proteins were determined as described previously [17]. All of the primers are shown in Table S1. The coding sequence was amplified with primers PLx1 and PLx2 (X: BbAtg22A-BbAtg22D) using cDNA as a template. All of the PCR experiments were performed according to the standard procedures. The resulting amplified band was cloned into pBMGS and fused with the 5 -end of the green fluorescent protein gene (GFP). The expression vector was transformed into the WT strain, and the candidate transformant was screened on the CZA with phosphinothricin (200 µg/mL). Fungal strains were grown in SDB medium (SDAY plate without agar) at 25 • C for 2 d, and the resulting mycelia were stained with 7-amino-4-chloromethylcoumarin (CMAC) indicating vacuoles or nile red indicating lipid droplets. The fluorescent signals in the mycelia were observed with a laser scanning confocal microscope (LSM 710, Carl Zeiss Microscopy GmbH, Jena, Germany).

Targeted Gene Disruption
All of the B. bassiana disruption mutants were generated using homologous replacement coupled with a fluorescence reporter [18]. All primers are included in Table S1. The up-and down-stream flanking regions of the indicated gene were amplified using the primer pairs P X 1/P X 2 and P X 3/P X 4, respectively. The resulting fragments were sequen-tially cloned into the XmaI/BamHI and XbaI/HpaI sites of p0380-bar using the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China), generating the gene disruption vector (p0380-bar-X). The resulting vector was transformed into the WT strain with the Agrobacterium-based transformation method, and the transformants were screened on CZA plates with phosphinothricin (200 µg/mL). To complement the gene loss, the full-length gene was amplified with the primer pair P X 5/P X 6, and the obtained fragment was inserted into the plasmid pPK2-NTC-GFP containing the nourseothricin-resistance gene [19]. For screening the complementation mutants, transformants were grown on a CZA plate supplemented with nourseothricin (50 µg/mL). All transformants were screened by PCR with the primer pair P X 7/P X 8, and the gene disruption mutant was further verified with the fluorescence-coupled double screening method.

Visualizing Autophagic Flux in Fungal Development
Fusion protein GFP-Atg8 (GA8) was used as a marker to track the autophagic process [4]. Plasmid p0380-GA8-sur was integrated into the wild type and four gene disruption mutant strains. To visualize autophagy in the aerial mycelia, conidia of the transgenic strain were inoculated on SDAY plates and cultured at 25 • C. The aerial mycelia were sampled at 3.5 days post-incubation. As for submerged mycelia, the conidia were inoculated into SDB (SDAY without agar) and cultured for 2 d at 25 • C. The mycelial samples were stained with CMAC and examined with a fluorescent microscope.
The conidial production was determined on a SDAY plate. Aliquots (100 µL of 10 7 conidia/mL) were evenly spread on agar plates and cultured for 7 d at 25 • C. Mycelial discs, 5 mm in diameter, were suspended in 0.02% Tween-80 solution. The conidial concentration was examined and used to calculate the conidial yield (conidial number per square centimeter). The fungal development under a submerged condition was assayed in SDB medium (SDAY without agar). The conidia were inoculated into SDB at the final concentration of 10 5 conidia/mL and cultivated for 3 d at 25 • C with constant shaking. The concentration of blastospores in the media was determined using microscopic counts, and the blastospore yield was shown as the number of spores per ml of culture.

Assays for Membrane Integrity
Nucleus staining with SYTOX Green was applied as previously described. Cells with green fluorescence means their cytomembrane is impaired [20]. The conidia suspension was inoculated onto the SDB and cultured for 2 d at 25 • C. The resultant mycelium were collected and stained with SYTOX Green for 10 min away from light. The green fluorescence was detected under a laser scanning confocal microscope (LSM 710, Carl Zeiss Microscopy GmbH, Jena, Germany).

Pathogenicity Assays
For pathogenicity tests, the last instar larvae of Galleria mellonella were used as the bioassay hosts, and each treatment included 30-35 larvae. The fungal strains were cultured on SDAY plates for 7 d at 25 • C, and the resultant conidia were used as infectious inocula. In the cuticle inoculation assay, insects were immersed in a conidial suspension (10 7 conidia/mL) for 10 s. In the intrahemocoel injection assay, conidial suspension (5 µL, 10 5 cells/mL) was injected into the host hemoceol. Tween-80 solution (0.02%) was used to suspend the conidia and also as the control in the assays. The daily-recorded mortality was used to calculate the median lethal time (LT 50 ) by the Kaplan-Meier method with a log-rank test for determining the difference between the paired survival trends.

qRT-PCR Assays
The transcriptional analyses for the genes were performed as reported previously [21]. The wild type strain was cultured on a SDAY plate, and the mycelia were sampled at the indicated time point. The total RNA was extracted from the mycelial samples with RNAiso TM Plus Reagent (TaKaRa, Dalian, China) according to the manufacturer's protocol. The cDNA was reverse transcribed using the PrimeScript ® RT reagent Kit (TaKaRa) and used as templates to perform the qRT-PCR reaction on a Mastercycler ® EP Realplex (Eppendorf, Hamburg, Germany) cycler. All primers are shown in Table S1. The relative expression level of each gene was calculated as the relative expression of different time points over 2 d using the 2 −∆∆CT method [22]. Fungal 18S rRNA was as an internal reference.

Statistical Analyses
One-way analysis of variance (ANOVA) was applied in the comparison of the phenotypic measurements between the disruptant and the wild type or complementation mutant, and the statistical significance was determined by a Tukey's honest significance test (Tukey's HSD). The analyses were performed with the software of GraphPad Prism 8 (GraphPad Software, Boston, MA, USA).
To further examine the biological roles of the four Atg22-like proteins, their disruption and complementation strains were constructed by homologous recombination and ectopic insertion strategies, respectively ( Figure S1).

Gene Expression and Cell Biology of Atg22-like Proteins
The transcriptional analyses indicated that the four BbAtg22 protein genes displayed dynamic expression profiles. The wild type strain was cultured on SDAY plates at 25 • C. The relative expression level of BbATG22D reached the maximal value at five days post incubation, and that of BbATG22C reached the maximal value at six days post-incubation ( Figure 2A). To further examine the biological roles of the four Atg22-like proteins, their disruption and complementation strains were constructed by homologous recombination and ectopic insertion strategies, respectively ( Figure S1).  The Atg22-like protein genes were fused to a GFP gene individually and transformed into the wild type strain. The fungal strains were cultured in SDB for two days. As illustrated in Figure 2B, BbAtg22A does not overlap with the vacuoles, which were labeled by CMAC (a vacuole specific dye), but completely overlaps with the lipid drop- The Atg22-like protein genes were fused to a GFP gene individually and transformed into the wild type strain. The fungal strains were cultured in SDB for two days. As illustrated in Figure 2B, BbAtg22A does not overlap with the vacuoles, which were labeled by CMAC (a vacuole specific dye), but completely overlaps with the lipid droplets labeled by Nile Red. The green fluorescence signals of BbAtg22B and BbAtg22C were completely consistent with those emitted by the CMAC (Figure 2C,D). BbAtg22D showed strong GFP signals along the cell peripheries and in the vacuoles. After removing the cell walls in the protoplasting experiment, the green fluorescence still remained in the cell periphery and vacuoles ( Figure 2E). These results suggest that these four Atg22-like proteins displayed the distinct sub-cellular locations.

Gene Expression and Cell Biology of Atg22-like Proteins
As illustrated in Figure 3, autophagic flux was indicated with the fusion protein GFP-Atg8. Green signals were co-localized with blue signals from the vacuole-specific dye in the wild type strain, as well as the four disruptants. This result indicates that the ablation of the four Atg22-like protein genes did not block the autophagic flux. As illustrated in Figure 3, autophagic flux was indicated with the fusion protein GFP-Atg8. Green signals were co-localized with blue signals from the vacuole-specific dye in the wild type strain, as well as the four disruptants. This result indicates that the ablation of the four Atg22-like protein genes did not block the autophagic flux.

Atg22-like Proteins Had Different Contributions to Cell Membrane Integrity
Cytomembrane integrity was determined by a SYTOX staining assay (Figure 4). Green fluorescent signals were observed in most cells of the ΔBbatg22A and ΔBbatg22D mutant strains; however, only less than 10% of the wild-type conidia were stained by this dye. The disruption of BbATG22B and BbATG22C did not significantly increase the percentage of stained cells. This indicated that the loss of BbAtg22A and BbAtg22D impaired the cytomembrane integrity.

Atg22-like Proteins Had Different Contributions to Cell Membrane Integrity
Cytomembrane integrity was determined by a SYTOX staining assay (Figure 4). Green fluorescent signals were observed in most cells of the ∆Bbatg22A and ∆Bbatg22D mutant strains; however, only less than 10% of the wild-type conidia were stained by this dye. The disruption of BbATG22B and BbATG22C did not significantly increase the percentage of stained cells. This indicated that the loss of BbAtg22A and BbAtg22D impaired the cytomembrane integrity.

Atg22-like Proteins Are Involved in Fungal Development
To determine the roles of the Atg22-like genes in nutrient utilization, th growth was evaluated on different carbon or nitrogen sources. After a seven-d bation at 25 °C, the disruption mutants showed no significant reduction in thei diameter when compared with the wild type ( Figure S3). Conidial production amined by spreading 100 µL conidial suspension (10 7 conidia/mL) on a SDAY p cubated at 25 °C. The microscopic examination indicated that the ΔBbatg22B, ΔB and ΔBbatg22D mutant strains produced significantly enlarged conidia-formin tures at 4 d post-incubation, and there was no significant difference between type and the ΔBbatg22A mutant strains ( Figure 6A).After a seven day incubation the conidial yields of ΔBbatg22A, ΔBbatg22B, ΔBbatg22C, and ΔBbatg22D were red 18.09%, 25.67%, 14.79%, and 24.94%, respectively, when compared with that of

Atg22-like Proteins Are Involved in Fungal Development
To determine the roles of the Atg22-like genes in nutrient utilization, the fungal growth was evaluated on different carbon or nitrogen sources. After a seven-day incubation at 25 • C, the disruption mutants showed no significant reduction in their colony diameter when compared with the wild type ( Figure S3). Conidial production was examined by spreading 100 µL conidial suspension (10 7 conidia/mL) on a SDAY plate, incubated at 25 • C. The microscopic examination indicated that the ∆Bbatg22B, ∆Bbatg22C and ∆Bbatg22D mutant strains produced significantly enlarged conidia-forming structures at 4 d post-incubation, and there was no significant difference between the wild type and the ∆Bbatg22A mutant strains ( Figure 6A).After a seven day incubation at 25 • C, the conidial yields of ∆Bbatg22A, ∆Bbatg22B, ∆Bbatg22C, and ∆Bbatg22D were reduced by 18.09%, 25.67%, 14.79%, and 24.94%, respectively, when compared with that of the wild type strain (8.57 ± 0.11 × 10 7 conidia/cm 2 ) ( Figure 6C). No significant differences were observed for the conidia-forming structures and conidial yield between the wild type and complemented strains. . Under submerged condition, conidial suspension were inoculated into SDB and cultured at 25 °C for 3 days, and then blastospore-forming structures were recorded as microscopic images (B). Conidial yield was examined at 7 dpi (C), and blastospore production was quantified at 3 dpi (D). Red and blue arrows indicate the conidium-and blastospore-forming structures, respectively. Scale bars: 10µm.Asterisks on the columns indicate a significant difference between the disruption mutant and the wild type or complemented strains [Tukey's honestly significant difference (HSD), p < 0.05]. Error bars: standard deviation.
After three days of incubation in the SDB liquid medium (Figure 6B), the blastospore-producing structures of four gene disruption mutants became shorter than those of the wild type. Except for ΔBbatg22C, the blastospore yields of the other three disruptants were significantly lower than that of the wild type (1.30 ± 0.06 × 10 8 spore/mL). The yield of ΔBbatg22A decreased to 1.00 ± 0.07 × 10 8 spore/mL; ΔBbatg22B decreased to 0.99 ± 0.10 × 10 8 spore/mL; ΔBbatg22D decreased to 1.06 ± 0.03 × 10 8 spore/mL ( Figure 6D).

Atg22-like Proteins Significantly Contributes to Fungal Virulence
Fungal proliferation in the host hemoceol was determined three days post infection ( Figure 7A). The wild type and complemented strains generated plenty of yeast-like . Under submerged condition, conidial suspension were inoculated into SDB and cultured at 25 • C for 3 days, and then blastospore-forming structures were recorded as microscopic images (B). Conidial yield was examined at 7 dpi (C), and blastospore production was quantified at 3 dpi (D). Red and blue arrows indicate the conidium-and blastospore-forming structures, respectively. Scale bars: 10µm.Asterisks on the columns indicate a significant difference between the disruption mutant and the wild type or complemented strains [Tukey's honestly significant difference (HSD), p < 0.05]. Error bars: standard deviation.

Atg22-like Proteins Significantly Contributes to Fungal Virulence
Fungal proliferation in the host hemoceol was determined three days post infection ( Figure 7A). The wild type and complemented strains generated plenty of yeast-like hyphal bodies, and there was no significant difference in the morphology among these strains. The yield of the hyphal body decreased by 25.53%, 46.81% and 41.48% in ∆Bbatg22A, ∆Bbatg22B, and ∆Bbatg22D, respectively, when compared with that of the wild type (7.88 ± 0.85 × 10 6 cells/mL). The yield of ∆Bbatg22C was slightly lower than that of the wild type, but it was not statistically significant ( Figure 7B). These results indicated that the loss of four Atg22-like proteins resulted in the impaired pathogenic growth in the host hemoceol.
hyphal bodies, and there was no significant difference in the morphology among the strains. The yield of the hyphal body decreased by 25.53%, 46.81% and 41.48% in Δ batg22A, ΔBbatg22B, and ΔBbatg22D, respectively, when compared with that of the wi type (7.88 ± 0.85 × 10 6 cells/mL). The yield of ΔBbatg22C was slightly lower than that the wild type, but it was not statistically significant ( Figure 7B). These results indicate that the loss of four Atg22-like proteins resulted in the impaired pathogenic growth in th host hemoceol. Conidial virulence for each strain against the greater wax moth Galleria mellonel was assayed by two methods of intrahemocoel injection and topical cuticle inoculatio As illustrated in Figure 7C, the survival percentage decreased with the incubation tim In two kinds of bioassay, excluding ΔBbatg22D, the other fungal strains could kill all th insect hosts. The LT50s for the wild type were 3.50 and 5.25 days in the injection and c Conidial virulence for each strain against the greater wax moth Galleria mellonella was assayed by two methods of intrahemocoel injection and topical cuticle inoculation. As illustrated in Figure 7C, the survival percentage decreased with the incubation time. In two kinds of bioassay, excluding ∆Bbatg22D, the other fungal strains could kill all the insect hosts. The LT 50 s for the wild type were 3.50 and 5.25 days in the injection and cuticle inoculation methods, respectively. In the intrahemocoel injection, the LT 50 values for the four disruptants (∆Bbatg22A, ∆Bbatg22B, ∆Bbatg22C and ∆Bbatg22D) were 4.00, 4.17, 4.17, 4.50, and 4.00 days, respectively. In the cuticle inoculation bioassay, the LT 50 values were prolonged by 1.42, 1.42, 0.75, and 0.75 days, respectively, when compared with that of the wild type ( Figure 7D). Apparently, the deletion of the Atg22-like protein genes greatly reduced the virulence of B. bassiana, indicating the essentiality of Atg22-like proteins for the fungal virulence.

Discussion
In yeast, Atg22 functions as a transporter to release the degradation products from the vacuoles into the cytoplasm when autophagy is activated [14]. In contrast to yeast, most filamentous fungi have one to four Atg22-like proteins [6]. In this study, four Atg22-like proteins were characterized in B. bassiana and display distinct sub-cellular localizations, in which BbAtg22A has the highest similarity to yeast Atg22, but their sub-cellular localizations were significantly different. These findings suggest that the Atg22 domain is not a determinant factor for the sub-cellular localization of Atg22-like proteins. Similar to the homolog in yeast, the four Atg22-like proteins do not contribute to biogenesis of autophagosomes, but play different roles in conidial germination, asexual development and virulence, as discussed below.
Fungal virulence is determinant for the potential of entomopathogenic fungi as biocontrol agents [23]. In B. bassiana, four Atg22-like proteins are involved in the fungal virulence. The involvement of the Atg22C homolog in fungal virulence has also been revealed in another B. bassiana strain by insertional mutagenesis [24]. Conidial germination is indispensable for the infection initiation caused by the entomopathogenic fungi [20]. As has been revealed, four proteins systematically contribute to conidial germination under the nutrient-limited condition. B. bassiana conidia accumulate a plethora of nutrients, including carbohydrates and lipids/fatty acids [20,25]. The surface of the host cuticle is an oligotrophic environment. B. bassiana conidia mobilize the reserved nutrients for germination and the invasive growth via autophagy [4,13]. Considering the different sub-cellular localizations, four Atg22-like proteins are involved in the nutrient mobilization in different manners in B. bassiana. BbAtg22A, localizing in the lipid droplets, might contribute to lipid metabolism for conidial germination. In yeast, Atg22 is an integral membrane protein localized on the vacuole membrane and recycles amino acids from the vacuoles under the starvation condition [14]. BbAtg22B and BbAtg22C localize in the vacuole and might function similarly to yeast Atg22 to release nutrients from the vacuole to the cytosol. BbAtg22D has an additional association with the cytomembrane, which indicates that this protein might contribute to assimilating nutrients from ambient environments. These findings suggest that Atg22-like proteins play comprehensive roles in nutrient acquisition.
In the host hemocoel, B. bassiana undergo dimorphic transmission and develop into yeast-like hyphal bodies (in vivo blastospore) [26]. The dimorphism between hyphal and yeast-like forms is essential for fungal pathogenesis [27]. Excluding BbAtg22C, the other three Atg22-like proteins in B. bassiana contribute to blastospore formation in the liquid media and host hemolymph. In B. bassiana, other genes related to lipid metabolism are associated with morphological transition, including acetyl-coenzyme A (CoA), synthetase 2 (Acs2) [28], and sterol carrier protein 2 (Scp2) [29]. BbAtg22A and BbAtg22D are required for cytomembrane integrity, which is critical for fungal virulence [20,21]. BbAcs2 contributes to maintaining the morphology of the lipid droplet and virulence in B. bassiana [28]. Thus, BbAtg22A and BbAtg22D are involved in fungal virulence in the intrahemocoel injection assay, which might be a combined effect of their roles in fungal dimorphism and membrane integrity. In addition, the four Atg22-like proteins perform different influences on conidiation. B. bassiana develop the conidiation process to generate conidia, which facilitates fungal dispersal and initiates the follow-up infection cycle [30]. Autophagy is an efficient transport system in which the nutrients are transferred through the tubular vacuoles during conidiation [31] and has been significantly linked to conidiation in B. bassiana [4,13]. In Fusarium oxysporum (a plant pathogenic pathogen), Atg22 is required for hyphal development and conidiation [32]. These results reinforce the idea that the nutrient assimilation is essential for conidiation in the filamentous fungi.

Conclusions
Taken together, the four Atg22-like proteins display distinct sub-cellular localizations and play different roles in fungal differentiation, the response to starvation stress, and virulence in B. bassiana. These findings suggest that filamentous fungi evolve more Atg22-like proteins to adapt to different ecological habitats. This study improves our understanding of the roles of Atg22-like protein in filamentous fungi beyond its conserved roles in autophagy.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof9020262/s1, Figure S1: Gene disruption and complementation in B. bassiana. Figure S2: Conidial germination of gene disruption mutants. Figure S3: Effects of the gene loss on vegetative growth of B. bassiana. Table S1: Primers used in this study.